Abstract Traditional strategies of vaccine development suffer from long-term and costly manufacture, and as a result, often fail to respond rapidly to newly emerging and reemerging infectious diseases. By contrast, messenger RNA (mRNA) is rising as a new technology platform to develop vaccines ?on demand? against viral pathogens, offering attractive advantages such as cell-free production, non-viral delivery, as well as simple, fast and cost- effective manufacture. Further improvement upon mRNA's stability and translation efficiency, understanding of their immune mechanisms, and evaluation of their protective efficacy will facilitate the development of next- generation mRNA vaccine technologies against diverse viral pathogens. Middle-East respiratory syndrome (MERS) coronavirus (MERS-CoV) is a highly pathogenic, emerging infectious virus posing a continuous threat to public health worldwide. There are currently no MERS vaccines approved for use in humans. MERS-CoV spike (S) protein, particularly its receptor-binding domain (RBD), is an important vaccine target. We have previously shown that MERS-CoV RBD contains a critical neutralizing domain capable of inducing strong cross-neutralizing antibodies and protecting human dipeptidyl peptidase 4-transgenic (hDPP4-Tg) mice against MERS-CoV infection with outstanding efficacy. However, production of subunit vaccines and other traditional vaccines has limitations, such as low expression and complex purification. To address these unmet challenges, we propose to rationally design and evaluate novel mRNA vaccines, using MERS-CoV as a model pathogen and MERS-CoV S protein as a target antigen. We hypothesize that with appropriate modification and optimization, MERS-CoV S protein RBD-based mRNA vaccines will demonstrate improved stability, increased translation efficiency, and enhanced immunogenicity in both mouse and non-human primates (NHP) models, with protective efficacy on par with the RBD-based subunit vaccine. The specific aims are to (1) rationally design MERS-CoV mRNA vaccines with improved stability and translation efficiency, (2) carefully optimize mRNA formulations and immunization regimens towards in-vivo evaluation of their immunogenicity and mode of action in wild-type mice, and (3) comprehensively evaluate protective efficacy of MERS-CoV mRNA vaccines and elucidate their protective mechanisms in hDPP4-Tg mice and NHPs. Of note, we will also examine the utility of new technologies such as microfluidics and next-generation sequencing (NGS) analysis of B-cell response in mRNA vaccine development and evaluation. The long-term goal is to develop a safe and effective mRNA vaccine that is able to (1) maintain sufficient quantity and quality suitable for industrial- scale production, and (2) meet the WHO Target Product Profiles for rapid onset of immunity in outbreak settings and long-term protection of people at high ongoing risk of MERS-CoV. Together, the proposed project will shed light on protective mechanisms of mRNA vaccines, and provide much-needed information and guidelines for developing mRNA vaccines against diverse viral pathogens with pandemic potential.